The interaction of specific DNA-binding proteins with their recognition sites in the eukaryotic chromosome is central to the mechanisms of gene regulation that occur in these cells. The techniques used to study these interactions rely primarily on methodology designed to detect preferential binding between proteins at various levels of purity from broken cell preparations and purified DNA sequences. We found (see Project ZOICP04986-09 LEC) that two tight-binding transcription factors are excluded from mouse mammary tumor virus (MMTV) chromatin in vivo. These findings suggest that the mere presence in the nucleus of the factors requisite to initiate transcription in vitro is not a sufficient condition for initiation to occur in vivo. Ultraviolet DNA-protein cross-linking has been utilized to address this problem. Using amplified minichromosomes based on the bovine papillomavirus (BPV) vector (see Project ZOICP05450-02 LEC), we have detected specific cross-links between minichromosome DNA two types of proteins, RNA polymerase II and histones, and have shown that loading of pol II on the minichromosome is hormone-dependent. Attempts are underway to determine if the interaction of single-copy regulatory proteins, such as the glucocorticoid receptor, with the template in vivo can be successfully monitored by these techniques.